Antiarrhythmic Effect Of Ethyl Acetate Extract From Chrysanthemum Morifolium Ramat On Rats

BACKGROUND:Acute myocardial infarction is one of the common cardiac emergencies,which will be effectively inhibited by restoring the occluded coronary blood perfusion as soon as possible. However,reperfusion itself may lead to accelerated and additional myocardial injury that is referred to as myocardial ischemia/reperfusion injury(MIRI).Reperfusion-induced arrhythmia is one of the severe MIRI which may contribute to sudden cardiac death.All antiarrhythmic drugs available in clinic exert their effects via changing some electrophysiological characteristics of myocardial cells and never been used chronically due to their great side effects.There is no long-term antiarrhythmic drug with high effect and little toxicity up to the present.The flower of Chrysanthemum morifolium(Ramat.)Tzvel(FCM)has been used as a folk medicine for sudorific,antidote and clearing away pathogenic heat,which is appointed to traditional medicine and healthy food by State Ministry of Health of the People's Republic of China.The previous studies found that the water extract of FCM increased coronary flow and contractility in the isolated rabbit and rat heart followed by ischemia-reperfusion.Moreover,clinical trials demonstrated that preparation of FCM has a beneficial effect on coronary heart disease,reduces the onset times of angina,the electrocardiographic abnormality and coronary perfusion shortage of coronary heart disease. However,there was no direct evidence to clarify the effect of FCM on arrhythmias induced by ischemia/reperfusion(I/R)or some cardiotoxic drugs such as aconitine.The objectives of the present study were as follows:(1)to investigate the effect of ethyl acetate extract from Chrysanthemum morifolium(Ramat.)Tzvel(CME)on experimental arrhythmias induced by aconitine in rats;(2)to investigate the effect of CME on isolated rat heart during I/R and its underlying mechanism;(3)to investigate the effect of CME on the action potentials of papillary muscle in rat right ventricle and its underlying mechanism by conventional glass microelectrode technique.METHODS:1.Experimental arrhythmia model induced by aconitineMale Sprague-Dawley(SD)rats of 240～280 g were used.They were anaesthetized by an intraperitoneal injection of urethane(1.2 g/kg)and electrocardiogram of leadâ…¡was examined.After the rats were treated with CME(1 mg/kg,4 mg/kg,8 mg/kg,i.v.)or saline (1.4 ml/kg,vehicle of CME)for 20 min,arrhythmias were induced by injecting aconitine (30μg/kg)in 5 sec,in which the number or duration of ventricular premature beats(VPB), ventricular tachycardia(VT)and ventricular fibrillation(VF)were examined.2.Quantification of arrhythmiasQuantification of arrhythmias was achieved by using scoring systems:score = (log₁₀VPBs)+ 2(log₁₀number of episodes of VT)+ 2[(log₁₀number of episodes of VF)+ (log₁₀total duration of VF)].3.Regional ischemia/reperfusion(I/R)model in isolated Langendorff-perfused rat heartMale SD rats were anesthetized by intraperitoneal injection of urethane(1.2 g/kg). Hearts were excised rapidly and placed in ice-cold Krebs-Henseleit(K-H)buffer,and then retrogradely perfused with a 95%O₂ + 5%CO₂ equilibrated K-H solution via the aorta at a constant pressure of 76 mmHg on a Langendorff apparatus.For hearts subjected to regional ischemia,a 5/0 silk suture was passed under the left coronary artery to form a snare.The artery was occluded for 30 min by pulling the snare to produce ischemia,while reperfusion was achieved by releasing it.All hearts were allowed to equilibrate for 20 min before any additional treatment.4.Measurement of electrophysiological parameters in myocardiumAll electrophysiological parameters,including ventricular fibrillation threshold(VFT), effective refractory period(ERP),and diastolic excitation threshold(DET),in rat heart were measured just before CME treatment,before and at 30 min of ischemia,and after 15 rain of reperfusion.Six stainless steel electrodes hooked at the epicardium:two at the left ventricle horizontally for recording,two at the right ventricle vertically for pacing,and the last two at the left ventricle vertically for measuring.The heart was paced by S1 at a rate of 5 Hz.Then an extrastimulus(S2)with a duration of 5 ms was applied every 8th of S1 with progressively increasing intensity until the stimulus can active the heart.DET was the lowest intensity capable of activating the heart in diastolic period.For the measurement of ERP,the heart was paced with S1 as described.An extrastimulus of twice the strength of DET and 5 ms duration was applied at the beginning of the diastolic interval following the excitation induced by S1.After 8 cycles,the interval between the S1 and S2 was progressively increased in steps of 2 ms until ventricular premature beats that caused by S2 was successful to capture.ERP was defined as the shortest interval between S1 and S2 that was able to cause VPB.VFT was determined with a train pulse electrical stimulation.In brief,the coupling interval(the interval between S2 and the R wave that was caused by last S1)is 30 ms.At each intensity 10 stimuli of 5 ms duration were applied with an inter-stimulus interval of 2 ms.The initial stimulus strength was 0.5 mA and subsequent stimulus were delivered at 0.2 mA increments.The lowest current required to produce ventricular fibrillation(VF)was VFT.VF was defined when more than 6 constant abnormal QRS waves after train stimulus appeared.5.Measurement of action potential in papillary muscle from the right ventricleMale SD rats were anesthetized by intraperitoneal injection of urethane(1.2 g/kg). Hearts were rapidly excised and transferred to a Petri dish containing K-H solution.The papillary muscles were excised from the right ventricle and mounted in a tissue bath.The preparations were superfused with a 95%O₂+5%CO₂ equilibrated K-H solution(pH 7.3-7.4) at a constant flow of 5 ml/min.The papillary muscles were stimulated through bipolar wires at 1 Hz with duration of 1 ms and intensity of twice threshold.Glass microelectrode (containing 3 mol/L KCl)with DC resistance 10-20 MΩwas used to record transmembrane potentials.The signals were recorded through a data acquisition system(RM6240BD, Chengdu,China).After stabilization for 1 h the papillary muscles were exposed to CME with increasing concentrations from 10 mg/L to 100 mg/L,15 min for each concentration. Five parameters including resting potential(RP),amplitude of action potential(APA), maximal velocity of phase 0 depolarization(V_max),action potential duration at 50%,90% of repolarization(APD₅₀,APD₉₀)were measured.Intracellular impalements were maintained at the same sites for all measurements.RESULTS:1.The effect of CME on aconitine-induced arrhythmiasPretreatment with CME(1 mg/kg,4 mg/kg,8 mg/kg)significantly decreased the number of VPB,VT and VF(P＜0.05 vs control group),and duration of VT and VF(P＜0.05 vs control group),delayed the occurrence of VPB.Quantification score of the CME group was lower than that in control group(P＜0.05).2.The effect of CME on electrophysiological parameters in myocardium2.1 The effect of CME on DET in myocardium after I/RPretreatment with CME did not affect the left ventricular DET after 30 min ischemia and 15 min reperfusion compared with control group(P＞0.05).There was also no difference in DET between ischemia and reperfusion period in all groups(P＞0.05).2.2 The effect of CME on ERP in myocardium after I/RLeft ventricular ERP was significantly shortened after 30 min ischemia(P＜0.05 vs pre-ischemia),and 15 min reperfusion did not significantly prolonged ERP.Pretreatment with low concentration of CME did not affect ERP in normal myocardium,but markedly prolonged ERP after 30 min ischemia.Pretreatment with both 50 mg/L and 100 mg/L CME significantly prolonged ERP in myocardium before ischemia,after 30 min ischemia and after 15 min reperfusion.2.3 The effect of CME on WT in myocardium after I/RPretreatment with CME did not alter VFT in normal myocardial.After 30 rain ischemia,myocardial VFT was decreased(P＜0.05 vs pre-ischemia).Pretreatment with both 50 mg/L and 100 mg/L CME significantly increased VFT after ischemia and reperfusion (P＜0.05 vs control group).However,pretreatment with 10 mg/L CME have no effect on VFT during I/R.3.The effect of CME on action potential in papillary muscle from the right ventriclePretreatment with 100 mg/L CME for 15 min significantly decreased V_maxin papillary muscle from the right ventricle(P＜0.05 vs control group).Pretreatment with CME also markedly prolonged APD₅₀and APD₉₀compared with control group(P＜0.05),but have no effect on RP and APA of the right ventricular papillary muscles.CONCLUSIONS:1.CME antagonizes the arrhythmogenic effect of aconitine.2.CME increases myocardial electrophysiological stability,and decreases the vulnerability of I/R myocardium.3.The mechanisms of antiarrhythmic and vulnerability lowing effects of CME may involve the inhibition of Na⁺ channels and its inactivation kinetics and the decrease of K⁺ efflux during repolarization.